Abstract Mammalian tissue culture models of gastrointestinal (GI) infection provide important tools for elucidating GI mechanisms of pathogenesis; however, such models currently lack methods to interrogate the role of human aging and biological rhythms in GI homeostatic responses to infection. In Project 3, we propose novel ?4- dimensional? experimental platforms to advance the study of three major GI pathogens: Helicobacter pylori (HP), Clostridium difficile (CD), and Shiga-toxin producing E. coli (STEC). Using inducible pluripotent stem cells (iPSC) isolated from human serum, we will generate two types of organoids: human gastric organoids (HGOs) and human colonic organoids (HCOs). Using GI epithelial tissue from patient biopsies, we will generate two types of enteroids: human ?gastroids? from stomach epithelium and human ?colonoids? from colon epithelium. Organoids and enteroids will be engineered and transduced with fluorescent and bioluminescent reporters of cell cycle and circadian rhythms. We will use these reporters for single-cell and population-level real-time analyses of epithelial turnover and circadian rhythms in: a) HGOs and gastroids infected with HP and b) HCOs and colonoids infected with CD toxins A and B and Shiga toxin. GI organoid and enteroid platforms will be derived from groups of infants, adolescents, young adults, and older adults. In Specific Aim 1, we will determine: a) which platform (organoid vs. enteroid) is the superior model for each pathogen, and b) the extent to which the age of the source influences response to infection. We hypothesize that HGOs and HCO's will be ?neonatal? like in their expression of cellular receptors to pathogens and toxins, whereas gastroids and colonoids will reflect the tissue maturity of the donor. We further hypothesize that enteroids derived from older adults will be highly susceptible to adverse outcomes from infection due to age-related impairments in GI stem cell regeneration. In Aim 2, we will determine whether: a) the time of day influences GI epithelial response to infection, b) GI pathogens disrupt epithelial timekeeping, and c) age-related perturbations in circadian rhythms modulate outcomes to GI infection. Collectively, these Specific Aims will bring together our NAMSED Projects and Cores for the common goal of developing robust ex vivo models of GI infection that lead to breakthrough therapies for the right patient at the right time.